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1.  Chemical management in fungicide sensivity of Mycosphaerella fijiensis collected from banana fields in México 
Brazilian Journal of Microbiology  2014;45(1):359-364.
The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L−1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L−1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.
PMCID: PMC4059323  PMID: 24948956
banana; fungicides; Mycosphaerella fijiensis; sensitivity; Sigatoka
2.  Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I 
Nature medicine  2013;19(6):753-759.
Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear1–6. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.
PMCID: PMC4019998  PMID: 23708290
3.  Clinically-relevant consecutive treatment with isoproterenol and adenosine protects the failing heart against ischaemia and reperfusion 
Consecutive treatment of normal heart with a high dose of isoproterenol and adenosine (Iso/Ade treatment), confers strong protection against ischaemia/reperfusion injury. In preparation for translation of this cardioprotective strategy into clinical practice during heart surgery, we further optimised conditions for this intervention using a clinically-relevant dose of Iso and determined its cardioprotective efficacy in hearts isolated from a model of surgically-induced heart failure.
Isolated Langendorff-perfused rat hearts were treated sequentially with 5 nM Iso and 30 μM Ade followed by different durations of washout prior to 30 min global ischaemia and 2 hrs reperfusion. Reperfusion injury was assessed by measuring haemodynamic function, lactate dehydrogenase (LDH) release and infarct size. Protein kinase C (PKC) activity and glycogen content were measured in hearts after the treatment. In a separate group of hearts, Cyclosporine A (CsA), a mitochondria permeability transition pore (MPTP) inhibitor, was added with Iso/Ade. Failing hearts extracted after 16 weeks of ligation of left coronary artery in 2 months old rats were also subjected to Iso/Ade treatment followed by ischaemia/reperfusion.
Recovery of the rate pressure product (RPP) in Iso/Ade-treated hearts was significantly higher than in controls. Thus in Iso/Ade treated hearts with 5 nM Iso and no washout period, RPP recovery was 76.3 ± 6.9% of initial value vs. 28.5 ± 5.2% in controls. This was associated with a 3 fold reduction in LDH release irrespective to the duration of the washout period. Hearts with no washout of the drugs (Ade) had least infarct size, highest PKC activity and also showed reduced glycogen content. Cardioprotection with CsA was not additive to the effect of Iso/Ade treatment. Iso/Ade treatment conferred significant protection to failing hearts. Thus, RPP recovery in failing hearts subjected to the treatment was 69.0 ± 16.3% while in Control hearts 19.7 ± 4.0%. LDH release in these hearts was also 3 fold lower compared to Control.
Consecutive Iso/Ade treatment of normal heart can be effective at clinically-relevant doses and this effect appears to be mediated by glycogen depletion and inhibition of MPTP. This intervention protects clinically relevant failing heart model making it a promising candidate for clinical use.
PMCID: PMC4045901  PMID: 24885907
Ischaemia/reperfusion; Cardioprotection; Isoproterenol; Adenosine; Heart failure; Mitochondria permeability transition pore
4.  Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes☆ 
L-type Ca channels (LTCC), which play a key role in cardiac excitation–contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation.
Graphical abstract
•Basal L type calcium current was reduced by interfering with caveolin-3 binding.•L type calcium current is tonically regulated by PKA phosphorylation.•Interfering with caveolin-3 binding reduced beta2 adrenergic stimulation of ICa.
PMCID: PMC3980375  PMID: 24412535
t-tubules; Ca; Phosphorylation; β2-Adrenoceptors
5.  A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes☆ 
Free Radical Biology & Medicine  2014;67(100):437-450.
The glycation of protein and nucleic acids that occurs as a consequence of hyperglycemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs after the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal, and disruption of mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear owing to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells, and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography–tandem mass spectrometry, enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycemia both in cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging.
•A mitochondria-targeted mass spectrometric probe, MitoG, has been developed to measure glyoxal and methylglyoxal.•Using MitoG we show that mitochondrial glyoxal and methylglyoxal can be measured in hyperglycemic cells.•MitoG can also be used in vivo to infer mitochondrial glyoxal and methylglyoxal production in a mouse model of type I diabetes.•These findings suggest that the accumulation of glyoxal and methylglyoxal within mitochondria may contribute to mitochondrial dysfunction in diabetes.
PMCID: PMC3978666  PMID: 24316194
Mitochondria; Exomarker; Methylglyoxal; Glyoxal; Hyperglycemia; MitoG; Free radicals
6.  Plant immunity. Methods and protocols 
Annals of Botany  2013;111(1):viii.
PMCID: PMC3523659
7.  Balloon Dilators for Labor Induction: a Historical Review 
A number of recent articles attribute the origin of the use of cervical balloon dilation in the induction of labor to either Barnes in the 1860s or Embrey and Mollison in the 1960s. This review examines the historical record and reveals that, based on current practice attribution should rather be made to two contemporaries of Barnes: the Storer and Mattei. More importantly, Storer’s warning about the rubber used in dilators was ignored, leading to decades of possibly unnecessary deaths following childbirth. To conduct this study key search terms for PubMed, Google Scholar and the website of the University of Ryerson were utilized as “Barnes”, “Woodman”, “balloon dilation”, “balloon catheter”, “foley”, “colpeurynter”, “cervix uteri” and “induction.” Subsequent analysis was done on downloaded articles using BibDesk.
PMCID: PMC3885146  PMID: 24427487
balloon dilation; cervical dilation; foley catheter; hydrostatic dilator; induction of labor
8.  Glycolytic ATP Fuels the Plasma Membrane Calcium Pump Critical for Pancreatic Cancer Cell Survival* 
The Journal of Biological Chemistry  2013;288(50):36007-36019.
Background: Pancreatic cancer cells exhibit up-regulated glycolysis.
Results: Inhibition of glycolysis, but not mitochondrial metabolism, induced ATP depletion, plasma membrane calcium pump (PMCA) inhibition, Ca2+ overload, and cell death.
Conclusion: Glycolytic ATP fuels the PMCA in pancreatic cancer.
Significance: Glycolytic regulation of the PMCA may represent a novel chemotherapeutic target for pancreatic cancer.
Pancreatic cancer is an aggressive cancer with poor prognosis and limited treatment options. Cancer cells rapidly proliferate and are resistant to cell death due, in part, to a shift from mitochondrial metabolism to glycolysis. We hypothesized that this shift is important in regulating cytosolic Ca2+ ([Ca2+]i), as the ATP-dependent plasma membrane Ca2+ ATPase (PMCA) is critical for maintaining low [Ca2+]i and thus cell survival. The present study aimed to determine the relative contribution of mitochondrial versus glycolytic ATP in fuelling the PMCA in human pancreatic cancer cells. We report that glycolytic inhibition induced profound ATP depletion, PMCA inhibition, [Ca2+]i overload, and cell death in PANC1 and MIA PaCa-2 cells. Conversely, inhibition of mitochondrial metabolism had no effect, suggesting that glycolytic ATP is critical for [Ca2+]i homeostasis and thus survival. Targeting the glycolytic regulation of the PMCA may, therefore, be an effective strategy for selectively killing pancreatic cancer while sparing healthy cells.
PMCID: PMC3861649  PMID: 24158437
ATP; Calcium ATPase; Calcium Signaling; Cell Death; Glycolysis; Metabolism; Pancreatic Cancer; PMCA; Warburg; Calcium Overload
9.  A simplified cervix model in response to induction balloon in pre-labour 
Induction of labour is poorly understood even though it is performed in 20% of births in the United States. One method of induction, the balloon dilator applied with traction to the interior os of the cervix, engages a softening process, permitting dilation and effacement to proceed until the beginning of active labour. The purpose of this work is to develop a simple model capable of reproducing the dilation and effacement effect in the presence of a balloon.
The cervix, anchored by the uterus and the endopelvic fascia was modelled in pre-labour. The spring-loaded, double sliding-joint, double pin-joint mechanism model was developed with a Modelica-compatible system, MapleSoft MapleSim 6.1, with a stiff Rosenbrock solver and 1E-4 absolute and relative tolerances. Total simulation time for pre-labour was seven hours and simulations ended at 4.50 cm dilation diameter and 2.25 cm effacement.
Three spring configurations were tested: one pin joint, one sliding joint and combined pin-joint-sliding-joint. Feedback, based on dilation speed modulated the spring values, permitting controlled dilation. Dilation diameter speed was maintained at 0.692 cm·hr-1 over the majority of the simulation time. In the sliding-joint-only mode the maximum spring constant value was 23800 N·m-1. In pin-joint-only the maximum spring constant value was 0.41 N·m·rad-1. With a sliding-joint-pin-joint pair the maximum spring constants are 2000 N·m-1 and 0.41 N·m·rad-1, respectively.
The model, a simplified one-quarter version of the cervix, is capable of maintaining near-constant dilation rates, similar to published clinical observations for pre-labour. Lowest spring constant values are achieved when two springs are used, but nearly identical tracking of dilation speed can be achieved with only a pin joint spring. Initial and final values for effacement and dilation also match published clinical observations. These results provide a framework for development of electro-mechanical phantoms for induction training, as well as dilator testing and development.
PMCID: PMC3850663  PMID: 24070547
Balloon dilator; Cervix; Pre-labour; Latent phase of labour; Labour induction; Dilation; Effacement
10.  Screw Placement Accuracy for Minimally Invasive Transforaminal Lumbar Interbody Fusion Surgery: A Study on 3-D Neuronavigation-Guided Surgery 
Global Spine Journal  2012;2(3):143-152.
Purpose The aim of this study was to assess the impact of 3-D navigation for pedicle screw placement accuracy in minimally invasive transverse lumbar interbody fusion (MIS-TLIF).
Methods A retrospective review of 52 patients who had MIS-TLIF assisted with 3D navigation is presented. Clinical outcomes were assessed with the Oswestry Disability Index (ODI), Visual Analog Scales (VAS), and MacNab scores. Radiographic outcomes were assessed using X-rays and thin-slice computed tomography.
Result The mean age was 56.5 years, and 172 screws were implanted with 16 pedicle breaches (91.0% accuracy rate). Radiographic fusion rate at a mean follow-up of 15.6 months was 87.23%. No revision surgeries were required. The mean improvement in the VAS back pain, VAS leg pain, and ODI at 11.3 months follow-up was 4.3, 4.5, and 26.8 points, respectively. At last follow-up the mean postoperative disc height gain was 4.92 mm and the mean postoperative disc angle gain was 2.79 degrees. At L5–S1 level, there was a significant correlation between a greater disc space height gain and a lower VAS leg score.
Conclusion Our data support that application of 3-D navigation in MIS-TLIF is associated with a high level of accuracy in the pedicle screw placement.
PMCID: PMC3864504  PMID: 24353961
minimally invasive spine surgery; transforaminal lumbar interbody fusion; 3D-NAV; neuronavigation; pedicle screw
11.  Nickel inhibits β-1 adrenoceptor mediated activation of cardiac CFTR chloride channels 
► We report the block of the β-adrenoceptor-activated cardiac CFTR Cl− current by Ni2+. ► Extracellular Ni2+ inhibits the current activated by β1-adrenoceptors in a concentration-dependent manner. ► The action of Ni2+ is insensitive to β2-blockade. ► Ni2+ does not affect the β-adrenoceptor-activated current from the intracellular side. ► The data are consistent with an action of Ni2+ at the β1-adrenoceptor from the external side.
Cardiac ventricular myocytes exhibit a protein kinase A-dependent Cl− current (ICl.PKA) mediated by the cystic fibrosis transmembrane conductance regulator (CFTR). There is conflicting evidence regarding the ability of the divalent cation nickel (Ni2+), which has been used widely in vitro in the study of other cardiac ionic conductances, to inhibit ICl.PKA. Here the action of Ni2+ on ICl.PKA activated by β-adrenergic stimulation has been elucidated. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. Externally applied Ni2+ blocked ICl.PKA activated by 1 μM isoprenaline with a log IC50 (M) of −4.107 ± 0.075 (IC50 = 78.1 μM) at +100 mV and −4.322 ± 0.107 (IC50 = 47.6 μM) at −100 mV. Thus, the block of ICl.PKA by Ni2+ was not strongly voltage dependent. Ni2+ applied internally via the patch-pipette was ineffective at inhibiting isoprenaline-activated ICl,PKA, but in the same experiments the current was suppressed by external Ni2+ application, indicative of an external site of Ni2+ action. In the presence of 1 μM atenolol isoprenaline was ineffective at activating ICl.PKA, but in the presence of the β2-adrenoceptor inhibitor ICI 118,551 isoprenaline still activated Ni2+-sensitive ICl.PKA. Collectively, these data demonstrate that Ni2+ ions produce marked inhibition of β1-adrenoceptor activated ventricular ICl.PKA at submillimolar [Ni2+]: an action that is likely to involve an interaction between Ni2+ and β1-adrenoceptors. The concentration-dependence for ICl.PKA inhibition seen here indicates the potential for confounding effects on ICl,PKA to occur even at comparatively low Ni2+ concentrations, when Ni2+ is used to study other cardiac ionic currents under conditions of β-adrenergic agonism.
PMCID: PMC3686155  PMID: 23376720
Rabbit cardiomyocytes; PKA-dependent Cl− current; CFTR; CFTR-inhibitor; Nickel; Ni2+
12.  Molecular Basis of Calcium-Sensitizing and Desensitizing Mutations of the Human Cardiac Troponin C Regulatory Domain: A Multi-Scale Simulation Study 
PLoS Computational Biology  2012;8(11):e1002777.
Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II binding site in the -free (apo) state closely resembled the -bound (holo) state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, , for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.
Author Summary
Muscle cells contract using a network of thread-like protein assemblies called myofilaments. Contraction is preceded by a signal that causes calcium to rush into the cell cytosol, where it can freely diffuse to and bind the myofilament proteins. Troponin C, a calcium sensor located on the thin filament, initiates and regulates the cascade of changes resulting in the generation of force by the thin and thick filaments comprising the myofilament lattice. In heart tissue, pathological conditions known as dilated and hypertrophic cardiomyopathies (DCM and HCM, respectively) are in part associated with abnormalities in the ability of the myofilaments to generate force at normal calcium concentrations. Manipulation of Troponin C calcium-binding through protein engineering and pharmaceutical intervention has thus attracted considerable attention as a therapeutic strategy for ameliorating these cardiac defects. In this study, we uncover a molecular basis of altered calcium handling for several engineered Troponin C variants, which provides further insight into tuning its control of myofilament contraction.
PMCID: PMC3510055  PMID: 23209387
13.  Acute desensitization of acetylcholine and endothelin-1 activated inward rectifier K+ current in myocytes from the cardiac atrioventricular node 
► ACh and ET-1 activate a K+ current in cardiac atrioventricular nodal cells. ► Tertiapin-Q sensitive IKACh activated via M2 receptors shows bi-exponential ‘fade’. ► ET-1 activates a similar current that also fades. ► The fade reflects desensitization rather than altered K+ ion driving force. ► Acetylcholine is able to cross-desensitize the AVN cell response to endothelin-1.
The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits ‘fade’ or desensitization in the AVN, as established for the heart’s primary pacemaker – the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1–1 μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at −120 mV; 1 μM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at −120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization.
PMCID: PMC3400056  PMID: 22683635
Acetylcholine (ACh); Atrioventricular node; AV node; AVN; Endothelin-1 (ET-1); GIRK; IKACh; Inward rectifier; Muscarinic potassium current; Tertiapin-Q
14.  Tissue-engineered total disc replacement: final outcomes of a murine caudal disc in vivo study  
Selected abstracts delivered at the 9th Annual AOSpine North America Fellows Forum
Consistent with EBSJ's commitment to fostering quality research, we are pleased to feature some of the most highly rated abstracts from the 9th Annual AOSpine North America Fellows Forum in Banff, Canada. Enhancing the quality of evidence in spine care means acknowledging and supporting the efforts of young researchers within our AOSpine North America network. We look forward to seeing more from these promising researchers in the future.
PMCID: PMC3506141  PMID: 23230409
15.  Modulation by Endothelin-1 of Spontaneous Activity and Membrane Currents of Atrioventricular Node Myocytes from the Rabbit Heart 
PLoS ONE  2012;7(3):e33448.
The atrioventricular node (AVN) is a key component of the cardiac pacemaker-conduction system. Although it is known that receptors for the peptide hormone endothelin-1 (ET-1) are expressed in the AVN, there is very little information available on the modulatory effects of ET-1 on AVN electrophysiology. This study characterises for the first time acute modulatory effects of ET-1 on AVN cellular electrophysiology.
Electrophysiological experiments were conducted in which recordings were made from rabbit isolated AVN cells at 35–37°C using the whole-cell patch clamp recording technique.
Application of ET-1 (10 nM) to spontaneously active AVN cells led rapidly (within ∼13 s) to membrane potential hyperpolarisation and cessation of spontaneous action potentials (APs). This effect was prevented by pre-application of the ETA receptor inhibitor BQ-123 (1 µM) and was not mimicked by the ETB receptor agonist IRL-1620 (300 nM). In whole-cell voltage-clamp experiments, ET-1 partially inhibited L-type calcium current (ICa,L) and rapid delayed rectifier K+ current (IKr), whilst it transiently activated the hyperpolarisation-activated current (If) at voltages negative to the pacemaking range, and activated an inwardly rectifying current that was inhibited by both tertiapin-Q (300 nM) and Ba2+ ions (2 mM); each of these effects was sensitive to ETA receptor inhibition. In cells exposed to tertiapin-Q, ET-1 application did not produce membrane potential hyperpolarisation or immediate cessation of spontaneous activity; instead, there was a progressive decline in AP amplitude and depolarisation of maximum diastolic potential.
Acutely applied ET-1 exerts a direct modulatory effect on AVN cell electrophysiology. The dominant effect of ET-1 in this study was activation of a tertiapin-Q sensitive inwardly rectifying K+ current via ETA receptors, which led rapidly to cell quiescence.
PMCID: PMC3315568  PMID: 22479400
16.  Selective Uncoupling of Individual Mitochondria within a Cell Using a Mitochondria-Targeted Photoactivated Protonophore 
Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the protonophore 2,4-dinitrophenol locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system. This also provides a proof of principle for the general temporally and spatially controlled release of bioactive molecules, pharmacophores, or toxins to mitochondria with tissue, cell, or mitochondrion specificity.
PMCID: PMC3260739  PMID: 22239373
We consider the design of an effective and reliable adaptive finite element method (AFEM) for the nonlinear Poisson-Boltzmann equation (PBE). We first examine the two-term regularization technique for the continuous problem recently proposed by Chen, Holst, and Xu based on the removal of the singular electrostatic potential inside biomolecules; this technique made possible the development of the first complete solution and approximation theory for the Poisson-Boltzmann equation, the first provably convergent discretization, and also allowed for the development of a provably convergent AFEM. However, in practical implementation, this two-term regularization exhibits numerical instability. Therefore, we examine a variation of this regularization technique which can be shown to be less susceptible to such instability. We establish a priori estimates and other basic results for the continuous regularized problem, as well as for Galerkin finite element approximations. We show that the new approach produces regularized continuous and discrete problems with the same mathematical advantages of the original regularization. We then design an AFEM scheme for the new regularized problem, and show that the resulting AFEM scheme is accurate and reliable, by proving a contraction result for the error. This result, which is one of the first results of this type for nonlinear elliptic problems, is based on using continuous and discrete a priori L∞ estimates to establish quasi-orthogonality. To provide a high-quality geometric model as input to the AFEM algorithm, we also describe a class of feature-preserving adaptive mesh generation algorithms designed specifically for constructing meshes of biomolecular structures, based on the intrinsic local structure tensor of the molecular surface. All of the algorithms described in the article are implemented in the Finite Element Toolkit (FETK), developed and maintained at UCSD. The stability advantages of the new regularization scheme are demonstrated with FETK through comparisons with the original regularization approach for a model problem. The convergence and accuracy of the overall AFEM algorithm is also illustrated by numerical approximation of electrostatic solvation energy for an insulin protein.
PMCID: PMC3178275  PMID: 21949541
Poisson-Boltzmann equation; semi-linear partial differential equations; super-critical nonlinearity; singularity; a priori L∞ estimates; existence; uniqueness; well-posedness; Galerkin methods; discrete a priori L∞ estimates; quasi-optimal a priori error estimates; adaptive finite methods; contraction; convergence; optimality; surface and volume mesh generation; mesh improvement and decimation
18.  Biological intervertebral disc replacement: an in vivo model and comparison of two surgical techniques to approach the rat caudal disc 
Study design: Prospective randomized animal study.
Objective: To determine a surgical technique for reproducible and functional intervertebral disc replacement in an orthotopic animal model.
Methods: The caudal 3/4 intervertebral disc (IVD) of the rat tail was approached by two surgical techniques: blunt dissection, stripping and retracting (Technique 1) or incising and repairing (Technique 2) the dorsal longitudinal tendons. The intervertebral disc was dissected and removed, and then either discarded or reinserted. Outcome measures were perioperative complications, spontaneous tail movement, 7T MRI (T1- and T2-sequences for measurement of disc space height (DSH) and disc hydration). Microcomputed tomographic imaging (micro CT) was additionally performed postmortem.
Results: No vascular injuries occurred and no systemic or local infections were observed over the course of 1 month. Tail movements were maintained. With tendon retraction (Technique 1) gross loss of DSH occurred with both discectomy and reinsertion. Tendon division (Technique 2) maintained DSH with IVD reinsertion but not without. The DSH was demonstrated on MRI measurement. A new scoring system to assess IVD appearances was described.
Conclusions: The rat tail model, with a tendon dividing surgical technique, can function as an orthotopic animal model for IVD research. Mechanical stimulation is maintained by preserved tail movements. 7T MRI is a feasible modality for longitudinal monitoring for the rat caudal disc.
PMCID: PMC3427968  PMID: 22956934
19.  Scalable representations of diseases in biomedical ontologies 
Journal of Biomedical Semantics  2011;2(Suppl 2):S6.
The realm of pathological entities can be subdivided into pathological dispositions, pathological processes, and pathological structures. The latter are the bearer of dispositions, which can then be realized by their manifestations — pathologic processes. Despite its ontological soundness, implementing this model via purpose-oriented domain ontologies will likely require considerable effort, both in ontology construction and maintenance, which constitutes a considerable problem for SNOMED CT, presently the largest biomedical ontology.
We describe an ontology design pattern which allows ontologists to make assertions that blur the distinctions between dispositions, processes, and structures until necessary. Based on the domain upper-level ontology BioTop, it permits ascriptions of location and participation in the definition of pathological phenomena even without an ontological commitment to a distinction between these three categories. An analysis of SNOMED CT revealed that numerous classes in the findings/disease hierarchy are ambiguous with respect to process vs. disposition. Here our proposed approach can easily be applied to create unambiguous classes. No ambiguities could be defined regarding the distinction of structure and non-structure classes, but here we have found problematic duplications.
We defend a judicious use of disjunctive, and therefore ambiguous, classes in biomedical ontologies during the process of ontology construction and in the practice of ontology application. The use of these classes is permitted to span across several top-level categories, provided it contributes to ontology simplification and supports the intended reasoning scenarios.
PMCID: PMC3102895  PMID: 21624161
20.  β-Adrenoceptor/PKA-stimulation, Na+–Ca2+ exchange and PKA-activated Cl− currents in rabbit cardiomyocytes: A conundrum 
Cell Calcium  2011;49(4):233-239.
Investigations into the functional modulation of the cardiac Na+–Ca2+ exchanger (NCX) by acute β-adrenoceptor/PKA stimulation have produced conflicting results. Here, we investigated (i) whether or not β-adrenoceptor activation/PKA stimulation activates current in rabbit cardiac myocytes under NCX-‘selective’ conditions and (ii) if so, whether a PKA-activated Cl−-current may contribute to the apparent modulation of NCX current (INCX). Whole-cell voltage-clamp experiments were conducted at 37 °C on rabbit ventricular and atrial myocytes. The β-adrenoceptor-activated currents both in NCX-‘selective’ and Cl−-selective recording conditions were found to be sensitive to 10 mM Ni2+. In contrast, the PKA-activated Cl− current was not sensitive to Ni2+, when it was activated downstream to the β-adrenoceptors using 10 μM forskolin (an adenylyl cyclase activator). When 10 μM forskolin was applied under NCX-selective recording conditions, the Ni2+-sensitive current did not differ between control and forskolin. These findings suggest that in rabbit myocytes: (a) a PKA-activated Cl− current contributes to the Ni2+-sensitive current activated via β-adrenoceptor stimulation under recording conditions previously considered selective for INCX; (b) downstream activation of PKA does not augment Ni2+-sensitive INCX, when this is measured under conditions where the Ni2+-sensitive PKA-activated Cl− current is not present.
PMCID: PMC3092849  PMID: 21439639
Cardiac myocyte; CFTR; NCX; Rabbit atrial myocyte; Rabbit ventricular myocyte; Whole-cell patch-clamp recording
21.  LFP spectral peaks in V1 cortex: network resonance and cortico-cortical feedback 
This paper is about how cortical recurrent interactions in primary visual cortex (V1) together with feedback from extrastriate cortex can account for spectral peaks in the V1 local field potential (LFP). Recent studies showed that visual stimulation enhances the γ-band (25–90 Hz) of the LFP power spectrum in macaque V1. The height and location of the γ-band peak in the LFP spectrum were correlated with visual stimulus size. Extensive spatial summation, possibly mediated by feedback connections from extrastriate cortex and long-range horizontal connections in V1, must play a crucial role in the size dependence of the LFP. To analyze stimulus-effects on the LFP of V1 cortex, we propose a network model for the visual cortex that includes two populations of V1 neurons, excitatory and inhibitory, and also includes feedback to V1 from extrastriate cortex. The neural network model for V1 was a resonant system. The model’s resonance frequency (ResF) was in the γ-band and varied up or down in frequency depending on cortical feedback. The model’s ResF shifted downward with stimulus size, as in the real cortex, because increased size recruited more activity in extrastriate cortex and V1 thereby causing stronger feedback. The model needed to have strong local recurrent inhibition within V1 to obtain ResFs that agree with cortical data. Network resonance as a consequence of recurrent excitation and inhibition appears to be a likely explanation for γ-band peaks in the LFP power spectrum of the primary visual cortex.
PMCID: PMC3050555  PMID: 19862612
Visual cortex; LPF; Oscillation; Gamma band; Resonance; Recurrent network
22.  Proteomic approaches to the characterization of protein thiol modification 
Protein cysteine residues are central to redox signaling and to protection against oxidative damage through their interactions with reactive oxygen and nitrogen species, and electrophiles. Although there is considerable evidence for a functional role for cysteine modifications, the identity and physiological significance of most protein thiol alterations are unknown. One way to identify candidate proteins involved in these processes is to utilize the proteomic methodologies that have been developed in recent years for the identification of proteins that undergo cysteine modification in response to redox signals or oxidative damage. These tools have proven effective in uncovering novel protein targets of redox modification and are important first steps that allow for a better understanding of how reactive molecules may contribute to signaling and damage. Here, we discuss a number of these approaches and their application to the identification of a variety of cysteine-centered redox modifications.
PMCID: PMC3087609  PMID: 21130020
23.  Pulsed lavage reduces the incidence of radiolucent lines under the tibial tray of Oxford unicompartmental knee arthroplasty 
International Orthopaedics  2009;33(6):1585-1590.
The aim of the study was to determine whether the incidence of radiolucencies can be reduced using pulsed lavage before cementing the tibia in unicompartmental knee arthroplasty (UKA). We prospectively studied a consecutive series of 112 cemented Oxford UKA in 100 patients in two centres. In group A (n = 56) pulsed lavage and in group B (n = 56) conventional syringe lavage was used to clean the cancellous bone. The same standardised cementing technique was applied in all cases. At a minimum follow-up of one year patients were evaluated clinically and screened radiographs were obtained. The cement bone interface under the tibial plateau was divided into four zones and evaluated for the presence of radiolucent lines. All radiographs were evaluated (n = 112), and radiolucencies in all four zones were found in two cases in group A (4%) and in 12 cases in group B (22%) (p = 0.0149). Cement penetration showed a median of 2.6 mm (group A) and 1.5 mm (group B) (p < 0.0001). We recommend the routine use of pulsed lavage in Oxford UKA to reduce the incidence of radiolucency and to improve long-term fixation.
PMCID: PMC2899174  PMID: 19219432
24.  Photodynamic therapy (PDT) in early central lung cancer: a treatment option for patients ineligible for surgical resection 
Thorax  2006;62(5):391-395.
To review the Yorkshire Laser Centre experience with bronchoscopic photodynamic therapy (PDT) in early central lung cancer in subjects not eligible for surgery and to discuss diagnostic problems and the indications for PDT in such cases.
Of 200 patients undergoing bronchoscopic PDT, 21 had early central lung cancer and were entered into a prospective study. Patients underwent standard investigations including white light bronchoscopy in all and autofluorescence bronchoscopy in 12 of the most recent cases. Indications for bronchoscopic PDT were recurrence/metachronous endobronchial lesions following previous treatment with curative intent in 10 patients (11 lesions), ineligibility for surgery because of poor cardiorespiratory function in 8 patients (9 lesions) and declined consent to operation in 3 patients. PDT consisted of intravenous administration of Photofrin 2 mg/kg followed by bronchoscopic illumination 24–48 h later.
29 treatments were performed in 21 patients (23 lesions). There was no procedure‐related or 30 day mortality. One patient developed mild skin photosensitivity. All patients expressed satisfaction with the treatment and had a complete response of variable duration. Six patients died at 3–103 months (mean 39.3), three of which were not as a result of cancer. Fifteen patients were alive at 12–82 months.
Bronchoscopic PDT in early central lung cancer can achieve long disease‐free survival and should be considered as a treatment option in those ineligible for resection. Autofluorescence bronchoscopy is a valuable complementary investigation for identification of synchronous lesions and accurate illumination in bronchoscopic PDT.
PMCID: PMC2117198  PMID: 17090572

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